Cellular signal transduction, i.e., the series of events leading from extracellular events to intracellular sequelae, is an aspect of cellular function in both normal and disease states. Numerous proteins that function as signal transducing molecules have been identified, including receptor and nonreceptor tyrosine kinases, phosphatases and other molecules with enzymatic or regulatory activities. These molecules generally demonstrate the capacity to associate specifically with other proteins to form a signaling complex that can alter cell activity.
Signaling proteins often contain domains of conserved sequence which serve as non-catalytic modules that direct protein-protein interactions during signal transduction. One such domain is the Src homology domain 2 (SH2), which is found in various combinations and locations in different proteins. For example, some members of the Src-family of tyrosine kinases, e.g., Abl, GRB2 and P13K, each contain one SH2 domain. Other tyrosine kinases, such as ZAP and Syk, contain two SH2 domains. The presence of multiple SH2 domains within a protein increases the variety of potential protein-protein interactions.
SH2 domains direct the association of specific proteins or protein domains by binding selectively and with specificity to protein sequences or motifs containing phosphotyrosine. For example, upon ligand binding, the PDGF beta-receptor dimerizes and autophosphorylates multiple tyrosine residues. This phosphorylation of tyrosine-containing protein motifs within the receptor triggers its physical association with SH2-containing proteins such as c-src, PLC-gamma, P13K and ras-GAP, forming a signaling complex. Other examples include the binding of the tandem SH2-containing protein Syk to tyrosine-phosphorylated motifs on the beta or gamma subunits of the IgE receptor, and the binding of the tandem SH2-containing protein ZAP-70 with tyrosine-phosphorylated motifs on subunits of the T cell receptor. Other protein receptors for tyrosine-phosphorylated protein domains are known and include the so-called phosphotyrosine binding domains ("PTBs") or phosphotyrosine interaction domains ("PIDs"). Many signaling pathways which play critical roles in disease processes are mediated by the binding of a phosphotyrosine-containing protein or protein domain with an SH2 or other protein receptor for a tyrosine-phosphorylated domain.
Pharmaceutical agents which interfere with the formation or stability of such signaling complexes may be used for precise intervention in these complex biological processes in order to treat or prevent the diseases or pathological effects mediated by such signaling. However, one common but significant problem when screening for pharmaceutically useful compounds that disrupt specific cellular events is deciphering their mechanism and specificity of action. Typically one searches for inhibition of a signaling cascade by measuring the effect of test compounds on an event several, if not many, steps downstream of a protein-protein interaction of particular interest. To the extent that a compound may exert its effects on any number of intermediate steps, the results of such experiments can be difficult to interpret.
A so-called "two-hybrid" interaction assay described by Song and Fields, Nature, 340:245-247 (1989) has been used to detect the interactions of a variety of molecules. See also, Fields et al, U.S. Pat. No. 5,283,173 (Feb. 1, 1994). The two-hybrid assay is based on the observation that transcription factors contain separable functional modules that direct either DNA binding or transcription activation. A DNA binding domain expressed in cells will bind to DNA but not activate transcription as it lacks a transcription activation domain. Conversely, a transcription activation domain alone will not affect transcription in the absence of directed and/or intimate interaction with DNA such as would be provided by a DNA-binding domain. However, if the DNA binding domain and the transcription activation domains are each expressed as part of separate fusion proteins, and the fusion proteins are capable of associating, the "two-hybrid" complex so formed represents a reconstituted transcription factor (see FIG. 1). Such a reconstituted transcription factor is capable of initiating transcription of a reporter gene (e.g., a gene for a conveniently detectable marker such as beta-galactosidase or alkaline phosphatase (SEAP) or a protein important for cell viability) located downstream of DNA binding sites recognized by the DNA-binding domain. The amount of reporter gene expression, i.e., the amount of gene product produced, will reflect the extent to which the fusion proteins complex with one another. Compounds that block the association of the fusion proteins will reduce reporter gene expression.
The two-hybrid assay approach has been used to identify presumed SH2-dependent protein-protein interactions using yeast [Xing, Z. et al., Mol. Biol. Cell, 5:413-421 (1994) and Osborne, M. A. et al., Biotechnol., 13:1474-1478 (1995)]. In those experiments, protein binding pairs, rather than inhibitors of such binding, were identified. The two hybrid approach has also been used to detect the inhibition of certain protein-protein interactions in yeast, but not for inhibition of protein-protein interactions involving phosphorylated ligands and their receptors [Chaudhuri, B. et al., FEBS Lett., 357: 221-226 (1995)]. While a yeast-based two-hybrid approach may be useful for other purposes, it would not lend itself to screening for drugs to inhibit phosphopeptide-mediated interactions in mammalian cells since the use of yeast cells would limit the researcher, or at least the "hits", to the subset of compounds which are able to penetrate the yeast cell wall. This artificial limitation would incorporate into the experimental design the risk of missing important new compounds capable of blocking key signaling interactions in mammalian cells, but which are unable to penetrate yeast cells.
There thus remains a need in the art for improved methods and compositions for identifying inhibitors of phosphopeptide-mediated protein-protein binding in a wide variety of host cells.